This application relates in general to the field of diagnostic microbiology. In particular, the invention relates to the species-specific detection of Aspergillus, Fusarium, Mucor, Penicillium, Rhizopus, Rhizomucor, Absidia, Cunninghamella, Pseudallescheria boydii (Scedosporium apiospermum), and Sporothrix species.
In recent years, chemotherapy for hematological malignancies, and high-dose corticosteroid treatment for organ transplant recipients, along with the spread of AIDS, have greatly increased the number of immunocompromised patients (1, 12, 14, 43). Saprophytic filamentous fungi, such as Aspergillus, Rhizopus, and Mucor species, found in the environment and considered to be of low virulence, are now responsible for an increasing number of infections in the immunocompromised host (17, 20, 43). In addition, these infections are often fulminant and rapidly fatal in immunocompromised patients (7, 11, 12, 20, 44). Morbidity and mortality is extremely high; for example, aspergillosis has a mortality rate of approximately 90% (8, 11).
To complicate matters, diagnosis is difficult and symptoms are often non-specific (18, 27, 29, 42, 44). Antibody-based tests can be unreliable due to the depressed or variable immune responses of immunocompromised patents (2, 9, 18, 46). Antigen detection tests developed to date have fallen short of the desired sensitivity (2, 9, 38). Radiographic evidence can be non-specific and inconclusive (5, 29, 36), although some progress in diagnosis has been made with the advent of computerized tomography (40). However, definitive diagnosis still requires either a positive blood or tissue culture or histopathological confirmation (3, 21). An added complication is that the invasive procedures necessary to obtain biopsy materials are often not recommended in thrombocytopenic patient populations (37, 41).
Even when cultures of blood, lung or rhinocerebral tissues are positive, morphological and biochemical identification of filamentous fungi can require several days for adequate growth and sporulation to occur, delaying targeted drug therapy. Some atypical isolates may never sporulate, making identification even more difficult (23). When histopathology is performed on tissue biopsy sections, the morphological similarities of the various filamentous fungi in tissue make differentiation difficult (16). Fluorescent antibody staining of histopathological tissue sections is not specific unless cross-reactive epitopes are absorbed out which can make the resultant antibody reactions weak (14, 19). Therapeutic choices vary (7, 41, 44) making a test to rapidly and specifically identify filamentous fungi urgently needed for the implementation of appropriately targeted therapy. Early and accurate diagnosis and treatment can decrease morbidity and increase the chances for patient survival (6, 27, 39). Furthermore, identification of filamentous fungi to at least the species level would be epidemiologically useful (24, 31, 43, 47).
PCR-based methods of detection, which show promise as rapid, sensitive means to diagnose infections, have been used in the identification of DNA from Candida species (13, 15, 30) and some other fungi, particularly Aspergillus species (31, 33, 45). However, most of these tests are only genus-specific (28, 38) or are directed to detect only single-copy genes (4, 35). Others have designed probes to detect multi-copy genes so as to increase test sensitivity (31, 33) but in doing so have lost test specificity because they have used highly conserved genes, which detect one or a few species but which are also plagued with cross-reactivities to human, fungal or even viral DNA (25, 31, 33).
Therefore, it is an object of the invention to provide improved materials and methods for detecting and differentiating Aspergillus and other filamentous fungal species in the clinical and laboratory settings.
The present invention relates to nucleic acids for detecting Aspergillus, Fusarium, Mucor, Penicillium, Rhizopus, Rhizomucor, Absidia, Cunninghamella, Pseudallescheria (Scedosporium), and Sporothrix species. Unique internal transcribed spacer 2 coding regions permit the development of probes specific for five different Aspergillus species, A. flavus, A. fumigatus, A. niger, A. terreus, and A. nidulans. The invention thereby provides methods for the species-specific detection and diagnosis of Aspergillus infection in a subject. In addition, species probes have been developed for three Fusarium, four Mucor, two Penicillium, five Rhizopus and one Rhizomucor species, as well as probes for Absidia corymbifera, Cunninghamella elegans, Pseudallescheria boydii (Scedosporium apiospermum), and Sporothrix schenckii. Generic probes for Aspergillus, Fusarium, and Mucor species have also been developed.
These and other objects, features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments and the appended claims.
This invention provides a simple, rapid, and useful method for differentiating filamentous fungal species from each other and from other medically important fungi. This invention enables a rapid, simple and useful method to isolate fungal DNA from host samples, and to apply the species- and genus-specific probes for the diagnosis of a disease. Ultimately, these probes can be used for in situ hybridization or in situ PCR diagnostics so that the morphology of host tissue, and microorganisms, remain intact.
The invention provides nucleic acids containing regions of specificity for five Aspergillus, three Fusarium, four Mucor, two Penicillium, five Rhizopus and one Rhizomucor species as well as probes for Absidia corymbifera, Cunninghamella elegans, Pseudallescheria boydii (Scedosporium apiospremum), and Sporothrix schenckii. These nucleic acids are from the internal transcribed spacer 2 (xe2x80x9cITS2xe2x80x9d) region of ribosomal deoxyribonucleic acid (rDNA) of the genome of the aforementioned filamentous fungi. The ITS2 region is located between the 5.8S rDNA region and the 28S rDNA region.
In particular, the invention provides nucleic acids from Aspergillus flavus (SEQ ID NO:1), Aspergillus fumigatus (SEQ ID NO:2), Aspergillus niger (SEQ ID NO:3), Aspergillus terreus (SEQ ID NO:4), Aspergillus nidulans (SEQ ID NO:5), Fusarium solani (SEQ ID NO:6), Fusarium moniliforme (SEQ ID NO:7), Mucor rouxii (SEQ ID NO:8), Mucor racemosus (SEQ ID NO:9), Mucor plumbeus (SEQ ID NO: 10), Mucor indicus (SEQ ID NO:11), Mucor circinilloides f. circinelloides (SEQ ID NO:12), Rhizopus oryzae (SEQ ID NO:13 and NO:14), Rhizopus microsportis (SEQ ID NO:15 and 16), Rhizopus circinans (SEQ ID NO:17 and 18). Rhizopus stolonifer (SEQ ID NO:19), Rhizomucor pusillus (SEQ ID NO:20), Absidia corymbifera (SEQ ID NO:21 and 22), Cunninghamella elegans (SEQ ID NO:23), Pseudallescheria boydii (teleomorph of Scedosporium apiospermum) (SEQ ID NO:24, 25, 26, and 27), Penicillium notatum (SEQ ID NO:28), and Sporothrix schenkii (SEQ ID NO:29). These sequences can be used to identify and distinguish the respective species of Aspergillus, Fusariunm, Mucor, Rhizopus, and Penicillium, and identify and distinguish these species from each other and from Absidia corymbifera, Cunninghamella elegans, Pseudallescheria boydii(Scedosporium apiospermum), and Sporothrix schenkii. 
Furthermore, the invention provides isolated nucleic acid probes derived from GenBank nucleic acid sequences (for Penicillium marneffei and Fusarium oxysporum only) or from the above nucleic acid sequences which may be used as species-specific identifiers of Aspergillus flavus (SEQ ID NO:30 and 31), Aspergillus fumigatus (SEQ ID NO:32), Aspergillus niger (SEQ ID NO:33), Aspergillus terreus (SEQ ID NO:34), Aspergillus nidulans (SEQ ID NO:35), Mucor rouxii (SEQ ID NO:36), Mucor plumbeus (SEQ ID NO:37), Mucor indicus (SEQ ID NO:38), Mucor circinilloides f. circinelloides (SEQ ID NO:39), Mucor racemosus (SEQ ID NO:40), Rhizopus oryzae (SEQ ID NO:41), Rhizopus circinans (SEQ ID NO:42), Rhizomucor pusillus (SEQ ID NO:43), Rhizopus stolonifer (SEQ ID NO:44), Pseudallescheria boydii (Scedosporium apiospermumn)(SEQ ID NO:45), Penicillium notatum (SEQ ID NO:46), Penicillium marneffei (SEQ ID NO:47 and 48), Fusarium moniliforme (SEQ ID NO:49), Fusarium oxysporum (SEQ ID NO:50), Fusarium solani (SEQ ID NO:51), Cunninghamella elegans (SEQ ID NO:52, 53, and 54), Absidia corymbifera (SEQ ID NO:55), Sporothrix schenkii (SEQ ID NO:56), and Rhizopus microsporus (SEQ ID NO:57). Such probes can be used to selectively hybridize with samples containing nucleic acids from species of Aspergillus, Fusarium, Mucor, Rhizopus (or Rhizomucor), Penicillium, or from Absidia corymbifera, Cunninghamella elegans, Pseudallescheria boydii (Scedosporium apiospermum), and Sporothrix schenkii. These fungi can be detected after polymerase chain reaction or ligase chain reaction amplification of fungal DNA and specific probing of amplified DNA with DNA probes labeled with digoxigenin, reacted with anti-digoxigenin antibodies labeled with horseradish peroxidase and a colorimetric substrate, for example. Additional probes can routinely be derived from the sequences given in SEQ ID NOs: 1-29, which are specific for the respective species. Therefore, the probes shown in SEQ ID NOs:30-57 are only provided as examples of the species-specific probes that can be derived from SEQ ID NOs: 1-29.
Generic probes for Aspergillus (SEQ ID NO:58), Fusarium, (SEQ ID NO:59) and Mucor (SEQ ID NO:60) species have also been developed to identify all members of their respective species which are listed above as well as an all-fungus biotinylated probe (SEQ ID NO:61) to capture all species-specific and generic probes listed above for their detection.
By xe2x80x9cisolatedxe2x80x9d is meant nucleic acid free from at least some of the components with which it naturally occurs. By xe2x80x9cselectivexe2x80x9d or xe2x80x9cselectivelyxe2x80x9d is meant a sequence which does not hybridize with other nucleic acids to prevent adequate determination of an Aspergillus, Fusarium, Mucor, Penicillium, Rhizopus or Rhizomucor genus or species or of Absidia corymbifera, Cunninghamella elegans, Pseudallescheria boydii (Scedosporium apiospermum), or Sporothrix schenckii species.
The hybridizing nucleic acid should have at least 70% complementarity with the segment of the nucleic acid to which it hybridizes. As used herein to describe nucleic acids, the term xe2x80x9cselectively hybridizesxe2x80x9d excludes the occasional randomly hybridizing nucleic acids and thus has the same meaning as xe2x80x9cspecifically hybridizingxe2x80x9d. The selectively hybridizing nucleic acids of the invention can have at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, and 99% complementarity with the segment of the sequence to which it hybridizes.
The invention contemplates sequences, probes and primers which selectively hybridize to the complementary, or opposite, strand of DNA as those specifically provided herein. Specific hybridization with nucleic acid can occur with minor modifications or substitutions in the nucleic acid, so long as functional species-specific or genus-specific hybridization capability is maintained. By xe2x80x9cprobexe2x80x9d is meant nucleic acid sequences that can be used as probes or primers for selective hybridization with complementary nucleic acid sequences for their detection or amplification, which probes can vary in length from about 5 to 100 nucleotides, or preferably from about 10 to 50 nucleotides, or most preferably about 18 nucleotides. The invention provides isolated nucleic acids that selectively hybridize with the species-specific nucleic acids under stringent conditions and should have at least 5 nucleotides complementary to the sequence of interest. See generally, Maniatis (26).
If used as primers, the invention provides compositions including at least two nucleic acids which hybridize with different regions so as to amplify a desired region. Depending on the length of the probe or primer, target region can range between 70% complementary bases and full complementarity and still hybridize under stringent conditions. For example, for the purpose of diagnosing the presence of the Aspergillus, the degree of complementarity between the hybridizing nucleic acid (probe or primer) and the sequence to which it hybridizes (e.g., Aspergillus DNA from a sample) is at least enough to distinguish hybridization with a nucleic acid from other yeasts and filamentous fungi. The invention provides examples of nucleic acids unique to each filamentous fungus in the listed sequences so that the degree of complementarity required to distinguish selectively hybridizing from nonselectively hybridizing nucleic acids under stringent conditions can be clearly determined for each nucleic acid.
Alternatively, the nucleic acid probes can be designed to have homology with nucleotide sequences present in more than one species of the fungi listed above. Such a nucleic acid probe can be used to selectively identify a group of species such as the generic probes listed for Aspergillus (SEQ ID NO:58), Fusarium (SEQ ID NO:59), and Mucor (SEQ ID NO:60) as well as all fungi listed (SEQ ID NO:61). Additionally, the invention provides that the nucleic acids can be used to differentiate the filamentous fungi listed in general from other filamentous fungi and yeasts, such as Candida species. Such a determination is clinically significant, since therapies for these infections differ.
The invention further provides methods of using the nucleic acids to detect and identify the presence of the filamentous fungi listed, or particular species thereof. The method involves the steps of obtaining a sample suspected of containing filamentous fungi. The sample may be taken from an individual, such as blood, saliva, lung lavage fluids, vaginal mucosa, tissues, etc., or taken from the environment. The filamentous fungal cells can then be lysed, and the DNA extracted and precipitated. The DNA is preferably amplified using universal primers derived from the internal transcribed spacer regions, 18S, 5.8S and 28S regions of the filamentous fungal rDNA. Examples of such universal primers are shown below as ITS1 (SEQ ID NO:62), ITS3 (SEQ ID NO:63), ITS4 (SEQ ID NO:64). Detection of filamentous fungal DNA is achieved by hybridizing the amplified DNA with a species-specific probe that selectively hybridizes with the DNA. Detection of hybridization is indicative of the presence of the particular genus (for generic probes) or species (for species probes) of filamentous fungus.
Preferably, detection of nucleic acid (e.g. probes or primers) hybridization can be facilitated by the use of detectable moieties. For example, the species-specific or generic probes can be labeled with digoxigenin, and an all-fungus probe, such as described in SEQ ID NO:61, can be labeled with biotin and used in a streptavidin-coated microtiter plate assay. Other detectable moieties include radioactive labeling, enzyme labeling, and fluorescent labeling, for example.